Enhancement of chemiluminescent assays

ABSTRACT

Chemiluminescent bioassays for the presence or concentration of an analyte in a sample use 1,2-dioxetanes as substrates for the enzyme of an enzyme complex that bind to the analyte. The chemiluminescence obtained from the decomposition of the dioxetane triggered by the enzyme through the formation of the corresponding 1,2-dioxetane oxyanion of the enzyme complex is enhanced by the addition of TBQ as an enhancement agent. Other polymeric quaternary onium salts can be used as enhancement agents in conjunction with enhancement additives which improve the ability of the enhancement agent to form hydrophobic regions in the aqueous sample, in which regions the 1,2-dioxetane oxyanion and its chemiluminescent decomposition products can be sequestered. A kit for performing such assays is also provided.

This application is a continuation-in-part of U.S. patent application Ser. No. 07/806,928, filed Dec. 12, 1991, now U.S. Pat. No. 5,330,900, itself a divisional application of U.S. patent application Ser. No 07/574,786, filed Aug. 30, 1990, issued as U.S. Pat. No. 5,112,960. The entire disclosure of U.S. Pat. No. 5,112,960 is incorporated herein by reference. This application is also a continuation-in-part application of U.S. patent application Ser. No. 07/959,531, filed Oct. 13, 1992, currently pending, the entire disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to the enhancement of chemiluminescent assays for analytes in a sample, generally a biological sample, based on the use of 1,2-dioxetanes as chemiluminescent substrates for enzyme-labeled targets or probes. The chemiluminescence of the dioxetane reporter molecule can be enhanced by the addition of a water-soluble quaternary polymer. Further enhancement can be achieved by additionally including in the sample an additive which improves the ability of the water-soluble quaternary polymer to sequester the dioxetane within hydrophobic regions formed by the water-soluble polymer thereby suppressing or avoiding water-induced light quenching reactions.

2. Background of the Prior Art

Chemiluminescent assays for the detection of the presence or concentration of an analyte in a sample, generally a biological sample, have received increasing attention in recent years as a fast, sensitive and easily read method of conducting bioassays. In such assays, a chemiluminescent compound is used as a reporter molecule, the reporter molecule chemiluminescing in response to the presence or the absence of the suspected analyte.

A wide variety of chemiluminescent compounds have been identified for use as reporter molecules. One class of compounds receiving particular attention is the 1,2-dioxetanes. 1,2-dioxetanes can be stabilized by the addition of a stabilizing group to at least one of the carbon molecules of the dioxetane ring. An exemplary stabilizing group is spiro-bound adamantane. Such dioxetanes can be further substituted at the other carbon position with an aryl moiety, preferably phenyl or naphthyl, the aryl moiety being substituted by an oxygen which is in turn bound to an enzyme-labile group. When contacted by an enzyme capable of cleaving the labile group, the oxyanion of the dioxetane is formed, leading to decomposition of the dioxetane and spontaneous chemiluminescence. A wide variety of such dioxetanes are disclosed in U.S. Pat. No. 5,112,960. That patent focuses on dioxetanes which bear a substituent on the adamantyl-stabilizing group, such as halo substituents, alkyl groups, alkoxy groups and the like. Such dioxetanes represent an advance over earlier-recognized dioxetanes, such as 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo]-3.3.1.1³,7 ]decan]-4-yl)phenyl phosphate, and in particular, the disodium salt thereof, generally identified as AMPPD. The chlorine-substituted counterpart, which converts the stabilizing adamantyl group from a passive group which allows the decomposition reaction to go forward to an active group which gives rise to enhanced chemiluminescence signal due to faster decomposition of the dioxetane anion, greater signal-to-noise values and better sensitivity, is referred to as CSPD (chloroadamant-2'-ylidene methoxyphenoxy-phosphorylated dioxetane). Other dioxetanes, such as the phenyloxy-β-D-galactopyranoside (AMPGD) are also well known, and can be used as reporter molecules. These dioxetanes, and their preparation, do not constitute an aspect of the invention herein, per se.

Assays employing these dioxetanes can include conventional assays, such as Southern, Northern and Western blot assays, DNA sequencing, ELISA, as well as other liquid phase and mixed phase assays. In general, the assay consists of binding the target, if present in the sample, with a complex bearing an enzyme capable of cleaving the enzyme labile group of the dioxetane. In DNA assays, the target is bound by a DNA probe with an enzyme covalently linked thereto, the probe being admixed with the sample immobilized on a membrane, to permit hybridization. Thereafter, excess enzyme complex is removed, and dioxetane added to the hybridized sample. If hybridization has occurred, the dioxetane will be triggered by the bound enzyme, leading to decomposition of the dioxetane, and chemiluminescence. In liquid phase assays, the enzyme is frequently conjugated or complexed with an antibody responsive to the target analyte, unbound complex being removed, and the dioxetane added, chemiluminescence being produced by the decomposition of the dioxetane triggered by the amount of enzyme present. In cases where the enzyme itself is the target analyte, the dioxetane need only be added to the sample. Again, a wide variety of assay modalities has been developed, as disclosed in U.S. Pat. No. 5,112,960, as well as U.S. Pat. No. 4,978,614.

It has been well known that light-quenching reactions will occur if the dioxetane decomposition goes forward in a protic solvent, such as water. As the samples suspected of containing or lacking the analyte in question are generally biological samples, these assays generally take place in an aqueous environment. The light-quenching reactions therefor may substantially reduce the chemiluminescence actually observed from the decomposition of the dioxetane. In assays involving low-level detections of particular analytes, such as nucleic acids, viral antibodies and other proteins, particularly those prepared in solution or in solution-solid phase systems, the reduced chemiluminescence observed, coupled with unavoidable background signals, may reduce the sensitivity of the assay such that extremely low levels cannot be detected. One method of addressing this problem is the addition of water-soluble macromolecules, which may include both natural and synthetic molecules, as is disclosed in detail in U.S. Pat. No. 5,145,772. The disclosure of this patent is incorporated herein, by reference. To similar effect, U.S. Pat. No. 4,978,614 addresses the addition of various water-soluble "enhancement" agents to the sample, although the patent speaks to the problem of suppressing non-specific binding in solid state assays. In U.S. Pat. No. 5,112,960, preferred water-soluble polymeric quaternary ammonium salts such as poly(vinylbenzyltrimethylammonium chloride) (TMQ) poly(vinyl-benzyltributylammonium chloride) (TBQ) and poly(vinylbenzyl-dimethylbenzylammonium chloride) (BDMQ) are identified as water-soluble polymeric quaternary ammonium salts which enhance chemiluminescence by the suppression of non-specific binding.

Notwithstanding the advances in technology addressed by these assays, it remains a goal of the industry to provide chemiluminescent assays of greater sensitivity, to determine the presence, concentration or both of an analyte in a sample which is generally biological, and therefor, in an assay in an aqueous environment. 1,2-dioxetane compounds have already been developed which show excellent potential as reporter molecules for such chemiluminescent assays. To be used in extremely low-level detection, however, and/or to improve reliability to provide for machine readability, it is necessary to further improve the enhancement of the chemiluminescence of the 1,2-dioxetane molecules produced in aqueous preparations.

SUMMARY OF THE INVENTION

Applicants' invention addresses the above goals in two embodiments. As noted above, it has been previously recognized that the addition of water-soluble polymeric ammonium salts to the aqueous sample improves or enhances chemiluminescence of 1,2-dioxetanes. Applicants have discovered that this enhancement is achieved, at least in part, through the formation of hydrophobic regions in which the dioxetane oxyanion is sequestered. Decomposition in these hydrophobic regions enhances chemiluminescence, because water-based light quenching reactions are suppressed. Among the recognized water-soluble quaternary polymer salts employed, TBQ provides unexpectedly superior enhancement, through this hydrophobic region-forming mechanism.

The chemiluminescent enhancement achieved by the addition of water-soluble polymeric quaternary polymer salts can be further improved by the inclusion, in the aqueous sample, of an additive, which improves the ability of the quaternary polymeric salt to sequester the dioxetane oxyanion and the resulting excited state emitter reporting molecule in a hydrophobic region. Thus, the combination of the polymeric quaternary salt and the additive, together, produce an increase in enhancement far beyond that produced separately by the addition of the polymeric quaternary salt, or the additive, which, when a surfactant or water-soluble polymer itself, may enhance chemiluminescence to a limited degree. The synergistic combination of the polymeric quaternary salt and additives gives enhancement effects making low-level, reliable detection possible even in aqueous samples through the use of 1,2-dioxetanes. The polymeric quaternary salts, coupled with the additives, are sufficiently powerful enhancers to show dramatic 4 and 5-fold increases at levels below 0.005 percent down to 0.001 percent. Increased signal, and improved signal/noise ratios are achieved by the addition of further amounts of the polymeric quaternary salt, the additive, or both, in amounts up to as large as 50 percent or more. In general, levels for both polymeric quaternary salt and additive can be preferably within the range of 0.05-25 percent, more preferably from 0.1-15 percent by weight.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph comparing the signal to noise ratio obtained by chemiluminescent decomposition of CSPD in the presence of the indicated enhancement agents.

FIG. 2 is a graph comparison of the signal (RLU) obtained from CSPD chemiluminescent decomposition in the presence of the indicated enhancement agents.

FIG. 3 is a graph comparison of the chemiluminescent (RLU) obtained from the decomposition of CSPD in the presence of zelec and/or TBQ under the indicated conditions.

FIG. 4 is a graph comparison of the signal/noise ratio obtained from the chemiluminescent decomposition of CSPD in the presence of zelec and/or TBQ under the indicated conditions.

DETAILED DESCRIPTION OF THE INVENTION

The assays and kits of this invention employ water-soluble chemiluminescent 1,2-dioxetanes. As noted above, these dioxetanes are well established in the art, and their identity and preparation does not constitute a novel aspect of this invention, per se. In general, any chemiluminescent dioxetane which exhibits sufficient stability in water to conduct the assay, and which may be caused to decompose and chemiluminesce by interaction with an enzyme, and cleavage, by the enzyme, of an enzyme labile group inducing the decomposition, can be used in connection with this invention. Typically, the 1,2-dioxetanes useful in this invention will have the general formula: ##STR1## R¹ is C₁ -C₂₀ alkyl or C₁₋₁₂ aryl or aralkyl; Y is phenyl or naphthyl;

R² is meta-substituted or non-conjugated on Y with respect to the dioxetane, and is OX, wherein;

X is an enzyme cleavable group which, when cleaved, leaves the dioxetane phenoxy or naphthoxy anion;

Z=H, Cl, other halogens, or alkoxy groups.

Preferred dioxetanes include AMPPD, and in particular, its disodium salt, as well as CSPD and its disodium salt. Methods of preparing these dioxetanes are disclosed in the aforereferenced commonly-assigned patents, as well as, e.g., U.S. Pat. No. 4,962,162, assigned to Wayne State University. The preparation, purification and isolation of the dioxetanes does not constitute a novel aspect of the invention disclosed and claimed herein per se.

The dioxetane is added to the sample which has been mixed with an enzyme complex which will bind to or otherwise co-act with the target analyte, if present in the sample. The dioxetane is therefore the substrate for the enzyme, the enzyme-catalyzed cleavage of the labile groups of the substrate from the body of the dioxetane resulting in the unstable oxyanion, and subsequent decomposition of the dioxetane. Where the target analyte is the enzyme itself, the dioxetane is added directly to the sample, either as drawn, or after preliminary purification to reduce turbidity. Where the target analyte is other than the trigger enzyme, the enzyme is complexed with a binding moiety, such as DNA probe or antibody, so as to bind to any target analyte present in the sample. The amount of chemiluminescence detected will be responsive both to the analyte in the sample, and the amount of analyte in the sample.

To enhance the chemiluminescent signal, and improve signal/noise ratio to permit discrimination between background signals and positive target-responsive signals at very low levels, a water-soluble enhancement agent is added to the sample prior or concomitant with the introduction of the dioxetane.

The enhancement agents of this invention, are based, in general, on polymeric onium salts, particularly quaternary salts based on phosphonium, sulfonium and, preferably, ammonium moieties. The polymers have the general formula I shown below: ##STR2##

In this formula each of R¹, R² and R³ can be a straight or branched chain unsubstituted alkyl group having from 1 to 20 carbon atoms, inclusive, e.g., methyl, ethyl, n-butyl, t-butyl, hexyl, or the like; a straight or branched chain alkyl group having from 1 to 20 carbon atoms, inclusive, substituted with one or more hydroxy, alkoxy, e.g., methoxy, ethoxy, benzyloxy or polyoxethylethoxy, aryloxy, e.g., phenoxy, amino or substituted amino, e.g., methylamino, amido, e.g., acetamido or ureido, e.g., phenyl ureido; or fluoroalkane or fluoroaryl, e.g., heptafluorobutyl, groups, an unsubstituted monocycloalkyl group having from 3 to 12 carbon ring carbon atoms, inclusive, e.g., cyclohexyl or cyclooctyl, a substituted monocycloalkyl group having from 3 to 12 ring carbon atoms, inclusive, substituted with one or more alkyl, alkoxy or fused benzo groups, e.g., methoxycyclohexyl or 1,2,3,4-tetrahydronaphthyl, a polycycloalkyl group having 2 or more fused rings, each having from 5 to 12 carbon atoms, inclusive, unsubstituted or substituted with one or more alkyl, alkoxy or aryl groups, e.g., 1-adamantyl or 3-phenyl-1-adamantyl, an aryl, alkaryl or aralkyl group having at least one ring and from 6 to 20 carbon atoms in toto, unsubstituted or substituted with one or more alkyl, aryl, fluorine or hydroxy groups, e.g., phenyl, naphthyl, pentafluorophenyl, ethylphenyl, benzyl, hydroxybenzyl, phenylbenzyl or dehydroabietyl; at least two of R₁, R₂ and R₃, together with the quaternary nitrogen atom to which they are bonded, can form a saturated or unsaturated, unsubstituted or substituted nitrogen-containing, nitrogen and oxygen-containing or nitrogen and sulfur-containing ring having from 3 to 5 carbon atoms, inclusive, and 1 to 3 heteroatoms, inclusive, and which may be benzoannulated, e.g., 1-pyridinium, 1-(3-alkyl or aralkyl)imidazolium, morpholino, alkyl morpholinium, alkylpiperidinium, N-acylpiperidinium, piperidino or acylpiperidino, benzoxazolium, benzthiazolium or benzamidazolium.

The symbol X⁻ represents a counterion which can include, alone or in combination, moieties such as halide, i.e., fluoride, chloride, bromide or iodide, sulfate, alkylsulfonate, e.g., methylsulfonate, arylsulfonate, e.g., p-toluenesulfonate, substituted arylsulfonate, e.g., anilinonaphthylenesulfonate (various isomers), diphenylanthracenesulfonate, perchlorate, alkanoate, e.g., acetate, arylcarboxylate, e.g., fluorescein or fluorescein derivatives, benzoheterocyclic arylcarboxylate, e.g., 7-diethylamino-4-cyanocoumarin-3-carboxylate, organic dianionssuch as p-terephthalate may also be represented by X⁻.

The symbol n represents a number such that the molecular weight of such poly(vinylbenzyl quaternary ammonium salts) will range from about 500 to about 500,000 (weight average), and preferably from about 20,000 to about 70,000, as determined by intrinsic viscosity or LALLS techniques.

Methods for the preparation of these polymers, related copolymers and the related starting materials where M is nitrogen are disclosed in G. D. Jones et al, Journal of Polymer Science, 25, 201, 1958; in U.S. Pat. Nos. 2,780,604; 3,178,396; 3,770,439; 4,308,335; 4,340,522; 4,424,326 and German Offenlegunsschrift 2,447,611.

The symbol M may also represent phosphorous or sulfur whereupon the corresponding sulfonium or phosphonium polymers have been described in the prior art: U.S. Pat. Nos. 3,236,820 and 3,065,272.

Methods of preparation of the polymers of this invention are set forth in the referenced U.S. Patents, as well as U.S. application Ser. No. 07/811,620 and do not constitute any aspect of this invention, per se.

Copolymers containing 2 or more different pendant onium groups may also be utilized in the invention described herein. ##STR3## The symbols X, M', R^(1'), R^(2'), R^(3') are as described above for X, M, R¹ -R³. The symbols Y and Z represent the mole fraction of the individual monomers comprising the copolymer. The symbols Y and Z may thus individually vary from 0.01 to 0.99, with the sum always equalling one.

As preferred moieties, M is N, and R¹ -R³ are individually, independently, alkyl, cycloalkyl, polycycloalkyl (e.g. adamantane) aralkyl or aryl, having 1 to 20 carbon atoms, unsubstituted or further substituted with hydroxyl, amino, amido, ureido groups, or combine to form via a spiro linkage to the M atom a heterocyclic (aromatic, aliphatic or mixed, optionally including other N, S or O heteroatoms) onium moiety.

Applicants have discovered that poly(vinylbenzyltributylammonium chloride), alone, gives unpredictably superior enhancement in combination with a chemiluminescent assay based on the use of a 1,2-dioxetane reporter molecule, as described above. In point of fact, the improvement in chemiluminescent signal obtained by the addition of TBQ, alone, was generally on the order of at least twice the improvement obtained through addition of the next best enhancement agent, BDMQ. For completeness, TBQ was also evaluated, alone, versus cetyltrimethylammonium bromide, (CTAB) another art recognized enhancement agent.

Evaluation of TBQ Alone as an Enhancement Agent

Protocol for the Evaluation of Various Enhancers

The enhancer samples were prepared from stock solutions in a 0.1 M diethanolamine, 1 mM MgCl₂, pH 9.5 buffer. CSPD or AMPPD was added to 0.5 ml of an enhancer solution (final dioxetane concentration. 0.4 mM). The background was recorded at room temperature in a Berthold 952T luminometer 10 and 60 minutes after the dioxetane addition. Subsequently, alkaline phosphatase was added (final enzyme concentration, 9.35×10⁻¹⁴ M), and the chemiluminescent signal was measured at 10, 30 and 60 (not for all samples) minutes as a 5 sec. integral.

All buffers contained 1 mM MgCl₂.

* * The half-time to plateau was determined as follows: 0.4 mM AMPPD in the desired buffer was preincubated at 37° C. or 30° C. and alkaline phosphatase was then added to each tube (final concentration 4.1×10⁻¹³ M). The tube was inserted into a Turner model 20-E Luminometer measuring in Turner Light Units (TLU) and the half-time to steady state light emission was calculated.

                  TABLE 1                                                          ______________________________________                                         HALF-LIFE OF AMPPD                                                             Effect of CTAB, TBQ, BDMQ, pH and Temperature                                  Half-Time to Plateau**                                                         pH    Buffer alone                                                                              + CTAB     + TBQ  + BDMQ                                      ______________________________________                                         30° C., 0.1 M Sodium Carbonate                                          9.0   3.66       17.5       3.48   4.50                                        9.6   2.07       9.8        2.79   2.62                                        10.0  1.62       7.4        2.17   2.15                                        30° C., 0.75 M Sodium Carbonate                                         9.0   4.19       33.5       6.03   4.84                                        9.6   2.12       19.3       4.15   2.82                                        10.0  1.67       13.3       2.84   2.27                                        37° C., 0.1 M Sodium Carbonate                                          9.0   2.37       10.1       2.53   2.52                                        9.6   1.34       5.36       1.65   1.48                                        10.0  1.06       4.06       1.29   1.28                                        37° C., 0.75 M Sodium Carbonate                                         9.0   2.37       22.0       3.33   2.81                                        9.6   1.29       9.6        2.29   1.62                                        10.0  0.95       6.5        1.61   1.33                                        ______________________________________                                    

                                      TABLE 2                                      __________________________________________________________________________     CHEMILUMINESCENCE OF AMPPD                                                     SIGNAL AND BACKGROUND                                                          Effect of CTAB, TBQ, BDMQ, pH and Temperature                                  Buffer Alone      Plus CTAB      Plus TBQ       Plus BDMQ                              Plus           Plus           Plus           Plus                         Back-                                                                               Alk Phos**                                                                               Back-                                                                               Alk Phos**                                                                               Back-                                                                               Alk Phos**                                                                               Back-                                                                               Alk Phos**                   ground*                                                                             TLU (time to                                                                             ground*                                                                             TLU (time to                                                                             ground*                                                                             TLU (time to                                                                             ground*                                                                             TLU (time to              pH TLU  max)   S/N                                                                               TLU  max)   S/N                                                                               TLU  max)   S/N                                                                               TLU  max)   S/N                __________________________________________________________________________     30° C., 0.1 M Sodium Carbonate                                          9.0                                                                               2.0  55.6 (31.6)                                                                           27.7                                                                              1.7  175.9 (93.0)                                                                          105.9                                                                             2.7  1110.8 (22.0)                                                                         405.4                                                                             2.4  875.0                                                                                 372.3)             9.6                                                                               2.0  50.7 (12.5)                                                                           25.0                                                                              1.6  114.1 (61.5)                                                                          69.6                                                                              3.8  1106.3 (33.8)                                                                         294.9                                                                             2.1  559.0                                                                                 267.4)             10.0                                                                              2.0  30.3 (10.8)                                                                           15.5                                                                              1.6   61.2 (42.4)                                                                          37.3                                                                              2.4   671.2 (18.0)                                                                         283.2                                                                             2.3  282.8                                                                                 123.0)             30° C., 0.75 M Sodium Carbonate                                         9.0                                                                               1.9  24.4 (30.7)                                                                           12.6                                                                              1.7   79.1 (88.5)                                                                          46.5                                                                              2.0   254.6 (43.7)                                                                         127.3                                                                             2.1  191.5                                                                                 93.00)             9.6                                                                               2.0  17.4 (15.4)                                                                           8.8                                                                               1.7   42.7 (115.3)                                                                         25.7                                                                              2.1   264.2 (29.7)                                                                         124.6                                                                             2.0  138.1                                                                                 70.86)             10.0                                                                              2.0  12.6 (9.8)                                                                            6.4                                                                               1.7   29.2 (80.3)                                                                          17.7                                                                              2.1   272.8 (19.3)                                                                         128.7                                                                             2.1  117.6                                                                                 57.19)             37° C., 0.1 M Sodium Carbonate                                          9.0                                                                               4.1  70.1 (17.2)                                                                           16.9                                                                              3.4  195.5 (53.7)                                                                          57.5                                                                              4.5  1687.4 (21.0)                                                                         371.7                                                                             4.4  900.0                                                                                 204.5)             9.6                                                                               4.2  61.6 (9.0)                                                                            14.7                                                                              3.3  132.2 (30.7)                                                                          40.1                                                                              4.4  1379.3 (11.4)                                                                         314.2                                                                             4.6  578.3                                                                                 125.4              10.0                                                                              4.1  41.0 (6.9)                                                                            10.0                                                                              3.3   76.6 (22.0)                                                                          23.2                                                                              4.7   824.3 (7.8)                                                                          176.9                                                                             4.3  305.8                                                                                 71.0)              37° C., 0.75 M Sodium Carbonate                                         9.0                                                                               4.1  27.3 (14.8)                                                                           6.6                                                                               3.5   93.4 (109.5)                                                                         26.6                                                                              4.2   410.7 (25.0)                                                                         97.3                                                                              4.3  293.1                                                                                 68.86)             9.6                                                                               4.2  19.8 (8.2)                                                                            4.8                                                                               3.4   51.7 (56.3)                                                                          15.2                                                                              4.4   416.0 (16.2)                                                                         94.8                                                                              4.3  230.8                                                                                 53.9)              10.0                                                                              4.3  15.0 (5.6)                                                                            3.5                                                                               3.4   30.3 (37.1)                                                                          9.0                                                                               4.2   402.2 (10.4)                                                                         95.8                                                                              4.2  173.5                                                                                 41.4)              __________________________________________________________________________

As can be seen from the foregoing enhancement analysis, TBQ offers sharp improvements in chemiluminescent enhancement, and elevated S/N, as compared with the dioxetane alone, or with other art-recognized enhancement agents. Thus, the addition to the sample of TBQ, alone, provides substantial enhancement of the chemiluminescent signal and S/N values obtained. In point of fact, Applicants have found that the addition of amounts as low as below 0.005 percent down to 0.001 percent, by weight, based on the sample volume, of TBQ results in an enhancement of the chemiluminescent signal 4-5 fold over the signal obtained from the dioxetane without any enhancer.

Addition of Enhancement Agent and Enchancement Additive

The polymeric quaternary salts that constitute the enhancement agents of this invention enhance chemiluminescence in a protic environment by forming hydrophobic regions in which the dioxetane moiety, resulting from the enzyme as the oxyanion, and the subsequently formed excited state are sequestered. By providing a hydrophobic region, light-quenching water reactions are reduced or avoided all together, resulting in an overall improvement of chemiluminescence. Applicants have discovered that use of an enhancement additive, in addition to the polymeric quaternary salt enhancement agents of the claimed invention, further enhances the observed chemiluminescent signal, and improves S/N values. The enhancement additives of the claimed invention show marked improvement, in connection with the enhancement agents, at very low levels. The addition of as much as 0.005 percent, by weight, based on the sample volume, of an enhancement additive further improves the strength of the chemiluminescent signal obtained by use of the enhancement agent, while not increasing the background levels. As with the enhancement agents, the additives can be used in amounts as high as practically useful. In general, amounts greater than 50 percent, by weight, based on the sample impede assay performance. Accordingly, inclusion of both the enhancement agent, and the enhancement additive, in amounts each of 0.005 percent, by weight, based on the sample volume, up to and including a total of 50 percent, by weight, based on the sample volume, of both, are within the scope of this invention. The total of agent and additive should not exceed 50 percent.

The function of the enhancement additive is to improve the ability of the enhancement agent to form hydrophobic regions in which the dioxetane oxyanion and the resulting emitter can be sequestered, permitting decomposition and chemiluminescence in the absence of water, and therefore, reducing light-quenching reactions caused thereby. The enhancement additives can be drawn from any of a wide variety of compounds. In general, the enhancement additives may, but need not necessarily, enhance the chemiluminescent signal obtained, although not nearly so strongly as the enhancement agents of this invention. Thus, the addition of conventional surfactants, largely detergents, improves the ability of the enhancement agent to form a hydrophobic region which is relatively stable. These surfactants may be cationic, anionic, zwitterionic or neutral. Another class of enhancement additives which when added to the solution appear to improve the ability of the enhancement agent to sequester the active dioxetane species, and in any event, lead to further enhancement of the chemiluminescent signal, include negatively charged salts. A third class of enhancement additives also active at very low concentrations are conventional solvents, including a wide variety of alcohols. Another conventional solvent, turpentine, is also useful in this role.

A fourth effective class of enhancement additives are non-quaternary water-soluble polymers, such as poly(2-ethyl-Z-oxazoline), (PolyOx). While these polymers themselves may induce limited enhancement of the chemiluminescent signal without increase in background noise, when added in conjunction with the polymeric quaternary onium salt enhancement agents of this invention, sharp improvements in the chemiluminescent signal observed are obtained.

To demonstrate the improvement obtained by the addition of an enhancement additive to the enhancement agent, buffered dioxetane solutions were provided with TBQ as well as sodium dodecylbenzenesulfonate, a surfactant, poly(2-ethyl-2-oxazoline) and octyl-β-glucoside. The results, set forth below in Tables 3 and 4, are obtained with CSPD and enhancer compositions, as indicated.

    ______________________________________                                         ABBREVIATIONS USED IN TABLES                                                   ______________________________________                                         TBQ    polyvinylbenzyltributylquaternary ammonium chloride                     SDBS   sodium dodecylbenzenesulfonate                                          PolyOx poly(2-ethyl-2-oxazoline), MW 500,000                                   OcGluc octyl-β-glucoside                                                  ______________________________________                                    

                                      TABLE 3                                      __________________________________________________________________________     SDBS/TBQ                                                                       TBA, mg/ml                                                                               SDBS, mg/ml                                                                            Noise, 10 min                                                                         SIGNAL 10'                                                                            SIGNAL 30'                                                                            SIGNAL 60'                                                                            S/N 10'                                                                              S/N                                                                                 S/N                   __________________________________________________________________________                                                              60'                   1 0.00    0.00    2.48   13829  14699  14007  56    59   57                    2 2.50    0.00    342    804466 1030372                                                                               952221 2352  3013 2784                  3 1.00    0.00    369    870920 1069979                                                                               1037251                                                                               2360  2900 2811                  4 0.50    0.00    327    632661 751145 691749 1935  2297 2115                  5 2.50    0.25    391    701661 1761807                                                                               2490243                                                                               1795  4506 6369                  6 1.00    0.10    403    956822 2332764                                                                               3060310                                                                               2374  5788 7594                  7 .50     0.05    399    577058 1410546                                                                               2000811                                                                               1446  3535 5015                  8 2.50    0.50    431    854086 2218583                                                                               3826962                                                                               1982  5148 8879                  9 1.00    0.20    416    932155 2245043                                                                               3513769                                                                               2241  5397 8447                  10                                                                               0.50    0.10    413    797925 1861389                                                                               2834250                                                                               1932  4507 6863                  11                                                                               1.00    0.30    424    752188 1908633                                                                               3161797                                                                               1774  4501 7457                  12                                                                               0.50    0.15    401    847070 2109424                                                                               3392238                                                                               2112  5260 8459                  13                                                                               1.00    0.40    575    829578 2217335                                                                               3785597                                                                               1443  3856 6584                  14                                                                               0.50    0.20    418    722581 1879004                                                                               3094497                                                                               1729  4495 7403                  15                                                                               1.00    0.50    512    886708 2310338                                                                               3885348                                                                               1732  4512 7589                  16                                                                               0.50    0.25    518    827050 2144813                                                                               3579020                                                                               1597  4141 6909                  17                                                                               0.75    0.50    492    707556 2012165                                                                               3549864                                                                               1438  4090 7215                  18                                                                               0.75    0.25    524    732565 1858120                                                                               2959896                                                                               1398  3546 5649                  19                                                                               0.75    0.10    489    859610 1968977                                                                               2895076                                                                               1758  4027 5920                  20                                                                               0.75    0.05    456    873340 1855213                                                                               2364623                                                                               1915  4068 5186                  21                                                                               0.50    0.50    254    9493   30130  56946  37    119  224                   22                                                                               0.50    0.25    531    667565 1959811                                                                               3045419                                                                               1257  3691 5735                  23                                                                               0.50    0.10    463    860156 2123923                                                                               3023727                                                                               1858  4587 6531                  24                                                                               0.50    0.05    421    863325 1873770                                                                               2390762                                                                               2051  4451 5679                  __________________________________________________________________________

                                      TABLE 4                                      __________________________________________________________________________     Polyox Detergents                                                                                                 Noise                                       TBQ,                               2nd Signal                                                                              Signal                                                                              Signal                                                                              S/N                                                                               S/N                                                                               S/N                mg/ml                              Run 10'  30'  60'  10'                                                                               30'                                                                               60'                __________________________________________________________________________     1 0.00                             236 14796                                                                               14981                                                                               15108                                                                               63 64 64                 2 1.00                             313 707879                                                                              846358                                                                              822417                                                                              2262                                                                              2704                                                                              2628               3 0.50                             290 734283                                                                              830370                                                                              797345                                                                              2532                                                                              2863                                                                              2749               4 0.00                                                                               20 mg/ml PolyOx              297 35058                                                                               39538                                                                               39785                                                                               118                                                                               133                                                                               134                5 1.00                                                                               20 mg/ml PolyOx              589 1302110                                                                             1706716                                                                             1710707                                                                             2228                                                                              2913                                                                              2910               6 0.50                                                                               "                            539 1430199                                                                             1892120                                                                             1851086                                                                             2652                                                                              3511                                                                              3432               7 0.50                                                                               "         5.0 mg/ml OcGluc   533 672726                                                                              862412                                                                              863895                                                                              1262                                                                              1618                                                                              1621               8 0.50                                                                               "         2.5 mg/ml OcGluc   432 894130                                                                              1083662                                                                             1047405                                                                             2070                                                                              2508                                                                              2425               9 0.50                                                                               "         1.0 mg/ml OcGluc   597 1244639                                                                             1585255                                                                             1503640                                                                             2085                                                                              2655                                                                              2519               10                                                                               0.50                                                                               "         0.5 mg/ml OcGluc   538 1333745                                                                             1679422                                                                             1607788                                                                             2479                                                                              3122                                                                              2988               11                                                                               0.50                                                                               1 mg/ml Tween 20                                                                         4.0 mg/ml OcGluc                                                                         0.5 mg/ml SDBS                                                                          255 16006                                                                               45080                                                                               84326                                                                               63 177                                                                               331                12                                                                               0.50                                                                               "         2.5 mg/ml OcGluc                                                                         0.25 mg/ml SDBS                                                                         515 669124                                                                              1676678                                                                             2761411                                                                             1299                                                                              3256                                                                              5362               13                                                                               0.50                                                                               "         1.0 mg/ml OcGluc                                                                         0.1 mg/ml SDBS                                                                          878 965179                                                                              2072592                                                                             2826307                                                                             1099                                                                              2361                                                                              3219               14                                                                               0.50                                                                               "         0.5 mg/ml OcGluc                                                                         0.05 mg/ml SDBS                                                                         770 916367                                                                              1818521                                                                             2278542                                                                             1190                                                                              2362                                                                              2959               15                                                                               1.00                                                                               "                   0.5 mg/ml SDBS                                                                          669 1145360                                                                             2693529                                                                             4466223                                                                             1712                                                                              4026                                                                              6676               16                                                                               1.00                                                                               "                   0.25 mg/ml SDBS                                                                         685 1087146                                                                             2341814                                                                             3439391                                                                             1587                                                                              3419                                                                              5021               17                                                                               1.00                                                                               "                   0.1 mg/ml SDBS                                                                          413 1457360                                                                             2768643                                                                             3541193                                                                             3529                                                                              6704                                                                              8574               18                                                                               1.00                                                                               "                   0.05 mg/ml SDBS                                                                         453 1091917                                                                             1889501                                                                             2266804                                                                             2410                                                                              4171                                                                              5004               __________________________________________________________________________

As is clearly reflected in the foregoing results, the addition of a variety of enhancement additives to the TBQ enhancement agent, in varying amounts, substantially improves chemiluminescent signal and S/N.

Further improvements in chemiluminescent signal and S/N can be obtained by combining two or more additives.

As reflected in the foregoing results (Table 4), although polyox yields some nominal improvement in an enhanced signal, as an enhancement additive in connection with TBQ, improved results are obtained. It should be noted that these results were obtained according to the protocol set forth above with regard to the assessment of TBQ, using AMPPD as a dioxetane.

Improvements in enhancement are obtained by the addition of TBQ, alone, or in particular, together with an enhancement additive, independent of the dioxetane identity, provided the dioxetane is a chemiluminescent one of the type suitable for use as described above. Thus, TBQ is used efficaciously in connection with both CSPD and AMPPD. Further, as noted, enhancement additives can be used in connection with TBQ to further improve both signal strength, and S/N values. One particular enhancement additive giving excellent results in connection with TBQ and CSPD is available from DuPont Corporation, under the trademark "Zelec", sold as an anti-static agent. The composition of Zelec is believed to be maintained as a trade secret, that includes turpentine, N-octylalcohol, water, isopropyl alcohol, sodium acetate, sodium oleyl sulfate, and TS compound or compounds identified as NJ Trade Secret Registry Number 00850201001-5400P. The Material Safety Data Sheet for the Zelec anti-stat is identified by No. 5909PP. As set forth herein below, combinations of Zelec and TBQ, or TBQ copolymers, give substantial improvements and results. AMPPD and CSPD alone, as well as together with BDMQ as an enhancement agent, or in the alternative, Zelec without an enhancement agent, are included for purposes of comparison.

                  TABLE 5                                                          ______________________________________                                                        10 Minutes                                                                               30 Minutes                                                           After Enzyme                                                                             After Enzyme                                          Dioxetane, Polymer                                                                          Noise   Signal   S/N  Signal S/N                                  ______________________________________                                         AMPPD        424     108,203  26   15,173 36                                   CSPS         281     16,350   58   16,581 59                                   AMPPD, BDMQ (1                                                                              334     148,273  444  273.770                                                                               820                                  mg/ml)                                                                         CSPD, TBQ (1 431     757,552  1,758                                                                               970,097                                                                               2,251                                mg/ml)                                                                         AMPPD, Zelec DP                                                                             529     12,596   24   26,622 50                                   (1 mg/ml)                                                                      AMPPD, Zelec DP                                                                             494     11,275   23   27,775 56                                   (2.5 mg/ml)                                                                    CSPD, Zelec DP (2.5                                                                         273     16,687   61   31,172 114                                  mg/ml)                                                                         AMPPD, BDMQ (1                                                                              725     363,815  502  834,997                                                                               1,152                                mg/ml), Zelec DP (1                                                            mg/ml)                                                                         AMPPD, BDMQ (1                                                                              900     469,637  522  1,334,310                                                                             1,483                                mg/ml), Zelec DP                                                               (2.5 mg/ml)                                                                    AMPPD, TBQ (1 mg/                                                                           1,077   431,674  401  1,311,966                                                                             1,218                                ml), Zelec DP (2.5                                                             mg/ml)                                                                         CSPD, BDMQ (1 mg/                                                                           428     1,119,735                                                                               2,616                                                                               2,560,207                                                                             5,982                                ml), Zelec DP (2.5                                                             mg/ml)                                                                         CSPD, TMQ (1 mg/                                                                            508     1,563,198                                                                               3,077                                                                               3,836,680                                                                             7,553                                ml), Zelec DP (2.5                                                             mg/ml)                                                                         ______________________________________                                          Buffer: 0.1 M diethanolamine, 1 mM MgCl.sub.2, pH 9.5                          Dioxetane concentration: 0.4 mM                                                Alkaline phosphatase concentration: 9,35 × 10.sup.-14 M                  Luminometer: Berthold 952T                                                     Data: 5 second RLU                                                       

Combinations of Enhancement Agent and Enhancement Additive

As noted previously, TBQ, alone, gives impressive improvements in observed chemiluminescence. TBQ in conjunction with an enhancement additive which improves the ability of TBQ to form hydrophobic regions in which chemiluminescent dioxetane species are sequestered further improves both chemiluminescent signal enhancement, and S/N ratio. It would of course be less desirable to improve the signal strength, if noise increased as well. The foregoing data clearly demonstrates that noise is maintained at a low level, when using commercially available dioxetanes, available from Tropix, Bedford, Mass. The improvements are far beyond those that could be expected by reason of the addition of TBQ alone, or the addition of the enhancement additive, alone, the additive occasionally having some, but generally modest, enhancement effects in the absence of the polymeric quaternary onium salt enhancement agent of the claimed invention.

To further demonstrate the improvements obtainable by using the polymeric quaternary onium salt enhancement agents of the claimed invention in combination with enhancement additives which can improve the ability of the enhancement agent to form hydrophobic regions in which chemiluminescent species are sequestered, a wide variety of combinations of enhancement agent, and one or more enhancement additives were prepared and evaluated for chemiluminescence. These enhancement agents are drawn from a wide variety of compound classes, including surfactants, negatively charged salts, conventional solvents, and water-soluble polymers. The results are reflected on the following pages.

The following method was used to evaluate various enhancer systems. Samples (0.5 ml) of each enhancer formulations were prepared from concentrated stock solutions. The buffer used was 0.1 M diethanolamine, 1 mM MgCl₂, pH 9.5. After the enhancer formulation was prepared, CSPD was added (final dioxetane concentration 0.04 mM). The background was then measured at room temperature in a Berthold 952T luminometer at approximately 10 minutes after substrate addition and at 1 hour. Next, alkaline phosphatase was added (final concentration, 9.35×10⁻¹⁴ M) and the chemiluminescent signal (5 second RLU) was measured at 10, 30 and 60 (sometimes) minutes.

    ______________________________________                                         Abbreviations used in tables:                                                  ______________________________________                                         AMPA-8   aminomethylated polyacrylamide, 8% solids                             AMPA-5   aminomethylated polyacrylamide, 5% solids                             AMPA-3   aminomethylated polyacrylamide, 3% solids                             BDMCAC   benzyldimethylcetyl ammonium chloride                                 BDMDAB   benzyldimethyldodecyl ammonium bromide                                BDMTDAC  benzyldimethyltetradecyl ammonium chloride                            BDMQ     poly(vinylbenzyldimethylbenzyl ammonium                                        chloride)                                                             BDMQ/TBQ BDMQ copolymerized with TBQ                                           Benz-PEI benzylated polyethylenimine (MW 70,000)                               BOP      benzotriazol-1-yloxytris-(dimethylamino)phos-                                  phonium hexafluorophosphate                                           CHAPS    (3-[(3-cholamidopropyl)-dimethylammonio]-1-                                    propanesulfonate)                                                     CHAPSO   (3-[(3-cholamidopropyl)-dimethylammonio]-2-                                    hydroxy-1-propanesulfonate)                                           CTAB     cetyl trimethyl ammonium bromide                                      DMQ-TEQ  poly(vinylbenzyldimethydodecyl ammonium                                        chloride) copolymerized with poly(vinylbenzyl-                                 triethylammonium chloride)                                            HDTBPB   hexadecyltributyl phosphonium bromide                                 OcGluc   octyl-β-glucoside                                                P-DMDMPC poly(1,1-dimethyl-3,5-dimethylene-piperidinium                                 chloride)                                                             PEI      polyethylenimine                                                      PEO      poly(ethylene oxide) (MW 7,000,000)                                   PO23LE   polyoxyethylene-23-lauryl ether                                       PolyOx   poly(2-ethyl-2-oxazoline)                                             PPG      polypropylene glycol                                                  PTHF     poly(tetrahydrofuran) (MW 250)                                        PVA      polyvinyl alcohol                                                     PVEE     poly(vinyl ethyl ether)                                               PVME     poly(vinyl methyl ether)                                              SBS      sodium benzyl suflate                                                 SDS      sodium dodecyl sulfate                                                SDBS     sodium dodecylbenzenesulfonate                                        TBQ      polyvinylbenzyltributyl ammonium chloride                             TBQ/THQ  TBQ copolymerized with poly(vinylbenzyltrihexyl                                ammonium chloride)                                                    TPP/TBQ  poly(vinylbenzyltriphenylphosphonium chloride)                                 copolymerizaed with TBQ                                               TPP/BDMQ poly(vinylbenzyltriphenylphosphonium chloride)                                 copolymerizaed with BDMQ                                              ______________________________________                                    

                                      TABLE 6                                      __________________________________________________________________________                               Signal         S/N                                   Additive      Concentration                                                                          Noise                                                                              10 min                                                                              30 min                                                                              60 min                                                                              10 min                                                                             30 min                                                                             60 min                        __________________________________________________________________________      1.                                                                               none               282 17417                                                                               17705     62  63                                 2.                                                                               BDMQ       1 mg/ml 536 369711                                                                              419173    690 782                                3.                                                                               TBQ        1 mg/ml 662 873852                                                                              1116942   1320                                                                               1687                               4.                                                                               Zelec DP   2.5 μl/ml                                                                           508 1563198                                                                             3836680   3077                                                                               7552                                 TBQ        1 mg/ml                                                           5.                                                                               polyvinyl alcohol                                                                         0.1 mg/ml                                                                              539 1004417                                                                             1281062   1863                                                                               2377                                 TBQ        1 mg/ml                                                           6.                                                                               isopropanol                                                                               1 mg/ml 574 962628                                                                              1271409   1677                                                                               2215                                 TBQ        1 mg/ml                                                           7.                                                                               2-octanol  10 mg/ml                                                                               568 1036203                                                                             1362463   1822                                                                               2399                                 TBQ        1 mg/ml                                                           8.                                                                               Zelec DP   2.5 μl/ml                                                                           927 837481                                                                              2586843   903 1860                                 SDS        1 mg/ml                                                             TBQ        1 mg/ml                                                           9.                                                                               Zelec DP   2.5 μl/ml                                                                           726 735959                                                                              2118539   1014                                                                               2918                                 SDS        1 mg/ml                                                             BDMQ       1 mg/ml                                                           10.                                                                              Zelec DP   2.5 μl/ml                                                                           853 594827                                                                              1586672   697 1860                                 Tween-20   5 mg/ml                                                             TBQ        1 mg/ml                                                           11.                                                                              Zelec DP   2.5 μl/ml                                                                           1129                                                                               545747                                                                              1289720   483 1142                                 Tween-20   0.01 mg/ml                                                          BDMQ       1 mg/ml                                                           12.                                                                              Zelec DP   2.5 μl/ml                                                                           1112                                                                               545295                                                                              1222221   490 1099                                 Tween-20   0.05 mg/ml                                                          BDMQ       1 mg/ml                                                           13.                                                                              Zelec DP   2.5 μl/ml                                                                           1338                                                                               957409                                                                              2375419   716 1775                                 Tween-20   0.01 mg/ml                                                          TBQ        1 mg/ml                                                           14.                                                                              Zelec DP   2.5 μl/ml                                                                           1097                                                                               969399                                                                              2345374   884 2138                                 Tween-20   0.05 mg/ml                                                          TBQ        1 mg/ml                                                           15.                                                                              TBQ/THQ    1 mg/ml 9997                                                                               5429086                                                                             8543357   543 855                                  (35 mole % THQ)                                                                CTAB       0.05 mg/ml                                                        16.                                                                              isopropanol                                                                               10 mg/ml                                                                               340 435740                                                                              533074    1282                                                                               1568                                 BDMQ       1 mg/ml                                                           17.                                                                              2-octanol  10 mg/ml                                                                               365 405753                                                                              510671    1112                                                                               1399                                 BDMQ       1 mg/ml                                                           18.                                                                              Zelec DP   3 μl/ml                                                                             10851                                                                              5605794                                                                             9637819   517 888                                  TBQ/THQ    1 mg/ml                                                             (35 mole % THQ)                                                              19.                                                                              AMPA-8     0.5 μl/ml                                                                           385 297926                                                                              400787    774 1041                                 BDMQ       1 mg/ml                                                           20.                                                                              AMPA-8     0.5 μl/ml                                                                           677 790317                                                                              1109404   1167                                                                               1639                                 TBQ        1 mg/ml                                                           21.                                                                              AMPA-5     0.5 μl/ml                                                                           398 530827                                                                              624527    1336                                                                               1569                                 BDMQ       1 mg/ml                                                           22.                                                                              AMPA-5     0.5 μl/ml                                                                           859 1006579                                                                             1289099   1172                                                                               1501                                 TBQ        1 mg/ml                                                           23.                                                                              AMPA-3     0.5 μl/ml                                                                           564 194022                                                                              251987    344 447                                  BDMQ       1 mg/ml                                                           24.                                                                              AMPA-3     0.5 μl/ml                                                                           796 757153                                                                              1021267   951 1283                                 TBQ        1 mg/ml                                                           25.                                                                              TPP/TBQ    2.5 mg/ml                                                                              407 9440 183054    232 450                                26.                                                                              TPP/BMQ    2.5 mg/ml                                                                              361 164641                                                                              201410    456 558                                27.                                                                              TBQ/THQ    1 mg/ml 4436                                                                               1501695                                                                             3853047   339 869                                  Tween-20   5 mg/ml                                                           28.                                                                              Zelec DP   2.5 μl/ml                                                                           8332                                                                               6693311                                                                             10215623  803 1226                                 TBQ/THQ    1 mg/ml                                                             (35 mole % THQ)                                                                Tween-20   1 mg/ml                                                           29.                                                                              Zelec DP   2.5 μl/ml                                                                           729 138425                                                                              332143    190 456                                  Tween-20   2.5 mg/ml                                                           TBQ/THQ    1 mg/ml                                                             (35 mole % THQ)                                                                CTAB       0.25 mg/ml                                                        30.                                                                              Zelec DP   2.5 μl/ml                                                                           402 633521                                                                              1675565   1576                                                                               4168                                 TritonX100 0.625 mg/ml                                                         TBQ        1 mg/ml                                                           31.                                                                              Zelec DP   2.5 μl/ml                                                                           381 673881                                                                              1714914   1769                                                                               4501                                 Tween-80   1.25 mg/ml                                                          TBQ        1 mg/ml                                                           32.                                                                              Benz-PEI   1 mg/ml 715 616720                                                                              1770606   863 2630                               33.                                                                              Lanoquat   1 mg/ml 495 56981                                                                               121737    115 246                                34.                                                                              TBQ/THQ    0.5 mg/ml                                                                              1574                                                                               5084709                                                                             6077275   3230                                                                               3861                                 (21 mole % THQ)                                                              35.                                                                              Benz-PEI   0.5 mg/ml                                                                              504 667698                                                                              151051    1325                                                                               3006                                 BDMQ       1 mg/ml                                                           36.                                                                              Lanoquat   0.5 mg/ml                                                                              401 222433                                                                              359929    555 898                                  BDMQ       1 mg/ml                                                           37.                                                                              Benz-PEI   1 mg/ml 858 745446                                                                              1774863   869 2069                                 TBQ        1 mg/ml                                                           38.                                                                              Lanoquat   0.5 mg/ml                                                                              570 600923                                                                              878859    1054                                                                               1542                                 TBQ        1 mg/ml                                                           39.                                                                              Hipofix DDD                                                                               1 mg/ml 418 346956                                                                              402685    830 963                                  BDMQ       1 mg/ml                                                           40.                                                                              Hipofix DDD                                                                               2.5 mg/ml                                                                              1040                                                                               760173                                                                              859718    731 822                                  TBQ        1 mg/ml                                                           41.                                                                              Hipofix DD-NF                                                                             0.5 mg/ml                                                                              343 345329                                                                              390776    1007                                                                               1139                                 BDMQ       1 mg/ml                                                           42.                                                                              Hipofix DD-NF                                                                             1 mg/ml 493 713154                                                                              854962    1447                                                                               1734                                 BDMQ       1 mg/ml                                                           43.                                                                              Hipofix 491                                                                               0.5 mg/ml                                                                              395 155086                                                                              199658    393 505                                  BDMQ       1 mg/ml                                                           44.                                                                              Hipofix 491                                                                               0.5 mg/ml                                                                              684 291815                                                                              366201    427 535                                  TBQ        1 mg/ml                                                           45.                                                                              Aerotex M-3                                                                               2.5 mg/ml                                                                              449 197957                                                                              233053    441 519                                  BDMQ       1 mg/ml                                                           46.                                                                              Aerotex M-3                                                                               5 mg/ml 791 492405                                                                              527229    623 723                                  TBQ        1 mg/ml                                                           47.                                                                              PVEE (lo mw)                                                                              1 mg/ml 568 2372633                                                                             378368    418 666                                  BDMQ       1 mg/ml                                                           48.                                                                              PVEE (lo mw)                                                                              1 mg/ml 655 730024                                                                              1151041   1115                                                                               1757                                 TBQ        1 mg/ml                                                           49.                                                                              Polyox (mw 50K)                                                                           1 mg/ml 574 342402                                                                              422461    597 740                                  BDMQ       1 mg/ml                                                           50.                                                                              Polyox (mw 50K)                                                                           1 mg/ml 699 1284452                                                                             1587003   1838                                                                               2270                                 TBQ        1 mg/ml                                                           51.                                                                              PVME       1.25 mg/ml                                                                             917 309446                                                                              371819    337 405                                  BDMQ       1 mg/ml                                                           52.                                                                              PVME       1.25 mg/ml                                                                             834 663814                                                                              839470    796 1007                                 TBQ        1 mg/ml                                                           53.                                                                              PTHF (mw 250)                                                                             2.5 mg/ml                                                                              700 265174                                                                              352224    379 503                                  BDMQ       1 mg/ml                                                           54.                                                                              PTHF (mw 250)                                                                             0.5 mg/ml                                                                              758 634639                                                                              851396    837 1123                                 TBQ        1 mg/ml                                                           55.                                                                              Polyox(mw 500K)                                                                           0.5 mg/ml                                                                              699 316630                                                                              385553    453 552                                  BDMQ       1 mg/ml                                                           56.                                                                              Polyox(mw 500K)                                                                           5 mg/ml 889 628735                                                                              822133    707 925                                  TBQ        1 mg/ml                                                           57.                                                                              PEO        1 mg/ml 670 220386                                                                              257387    329 384                                  BDMQ       1 mg/ml                                                           58.                                                                              PEO        1 mg.ml 776 541518                                                                              695872    698 897                                  TBQ        1 mg/ml                                                           59.                                                                              CelquatH100                                                                               0.5 mg/ml                                                                              406 284794                                                                              353576    701 871                                  BDMQ       1 mg/ml 406 284794                                                                              353576    701 871                                60.                                                                              CelquatH100                                                                               0.5 mg/ml                                                                              719 734280                                                                              970599    1021                                                                               1350                                 TBQ        1 mg/ml                                                           61.                                                                              Celquat L200                                                                              0.5 mg/ml                                                                              499 268280                                                                              326627    537 655                                  BDMQ       1 mg/ml                                                           62.                                                                              Celquat L200                                                                              0.5 mg/ml                                                                              969 722551                                                                              928806    746 959                                  TBQ        1 mg/ml                                                           63.                                                                              CelquatSC240                                                                              0.5 mg/ml                                                                              530 317199                                                                              382101    598 721                                             1 mg/ml                                                           64.                                                                              CelquatSC240                                                                              1 mg/ml 804 811714                                                                              1030836   1010                                                                               1282                                 TBQ        1 mg/ml                                                           65.                                                                              Pluronic 122                                                                              0.5 mg/ml                                                                              608 47854                                                                               117319    79  193                                  BDMQ       1 mg/ml                                                           66.                                                                              Pluronic 122                                                                              1 mg/ml 746 66584                                                                               181683    89  244                                  TBQ        1 mg/ml                                                           67.                                                                              Pluronic 123                                                                              0.5 mg/ml                                                                              648 69686                                                                               150161    108 232                                  BDMQ       1 mg/ml                                                           68.                                                                              Pluronic 123                                                                              0.5 mg/ml                                                                              1453                                                                               178632                                                                              386930    123 266                                  TBQ        1 mg/ml                                                           69.                                                                              Pluronic150R1                                                                             0.5 mg/ml                                                                              896 156254                                                                              173414    174 194                                  BDMQ       1 mg/ml                                                           70.                                                                              Pluronic150R1                                                                             0.5 mg/ml                                                                              1996                                                                               285940                                                                              324257    143 162                                  TBQ        1 mg/ml                                                           71.                                                                              CHAPS      0.5 mg/ml                                                                              210 14326                                                                               42519     68  202                                72.                                                                              CHAPSO     0.5 mg/ml                                                                              218 15119                                                                               42489     69  195                                73.                                                                              Zelec DP   2.25 μl/ml                                                                          638 741248                                                                              1616666   1162                                                                               2534                                 CHAPS      1 mg/ml                                                             BDMQ       1 mg/ml                                                           74.                                                                              Zelec DP   2.25 μl/ml                                                                          858 1146672                                                                             2754178   1336                                                                               3210                                 CHAPS      1 mg/ml                                                             TBQ        1 mg/ml                                                           75.                                                                              Zelec DP   2.25 μl/ml                                                                          555 718553                                                                              1530910   1295                                                                               2758                                 CHAPSO     1 mg/ml                                                             BDMQ       1 mg/ml                                                           76.                                                                              Zelec DP   2.25 μl/ml                                                                          900 1090555                                                                             2453191   1212                                                                               2726                                 CHAPSO     1 mg/ml                                                             TBQ        1 mg/ml                                                           77.                                                                              Polybrene  5 mg/ml 413 19740                                                                               24261     48  59                                   CHAPS      5 mg/ml                                                           78.                                                                              Polybrene  5 mg/ml 2853                                                                               486059                                                                              705032    170 247                                  CHAPS      5 mg/ml                                                             BDMQ       1 mg/ml                                                           79.                                                                              Polybrene  1 mg/ml 5982                                                                               683646                                                                              839733    114 140                                  CHAPS      1 mg/ml                                                             BDMQ       1 mg/ml                                                           80.                                                                              Polybrene  5 mg/ml 6348                                                                               832574                                                                              1239584   131 195                                  CHAPSO     5 mg/ml                                                             BDMQ       1 mg/ml                                                           81.                                                                              Polybrene  0.5 mg/ml                                                                              6441                                                                               736510                                                                              897356    114 139                                  CHAPSO     0.5 mg/ml                                                           BDMQ       1 mg/ml                                                           82.                                                                              BenzethoniumCl                                                                            0.5 mg/ml                                                                              692 34183                                                                               81211     49  117                                83.                                                                              BDMQ/TBQ   1 mg/ml 568 742480                                                                              859880    1307                                                                               1514                                 (20% BDMQ)                                                                   84.                                                                              BDMQ/TBQ   1 mg/ml 740 927108                                                                              1113083   1253                                                                               1504                                 (10% BDMQ)                                                                   85.                                                                              Zelec DP   2.5 μl/ml                                                                           1910                                                                               899187                                                                              1768511   471 926                                  BDMQ/TBQ   1 mg/ml                                                             (20% BDMQ)                                                                   86.                                                                              Zelec DP   2.5 μl/ml                                                                           8392                                                                               1057422                                                                             2177684   126 259                                  BDMQ/TBQ   1 mg/ml                                                             (10% BDMQ)                                                                   87.                                                                              Avitex ML  0.5 mg/ml                                                                              1051                                                                               142640                                                                              298297    136 284                                  BDMQ       1 mg/ml                                                           88.                                                                              Avitex ML  0.5 mg/ml                                                                              1318                                                                               466655                                                                              866897    354 658                                  TBQ        1 mg/ml                                                           89.                                                                              Polyox(500K)                                                                              0.5 mg/ml                                                                              1730                                                                               862023                                                                              1239458   498 716                                  CTAB       0.01 mg/ml                                                          TBQ        1 mg/ml                                                           90.                                                                              Polyox(50K)                                                                               0.5 mg/ml                                                                              952 872307                                                                              1183521   916 1243                                 CTAB       0.01 mg/ml                                                          TBQ        1 mg/ml                                                           91.                                                                              Agefloc B50                                                                               0.5 mg/ml                                                                              952 872307                                                                              1183521   1961                                                                               2262                                 TBQ        1 mg/ml                                                           92.                                                                              Agefloc B50                                                                               0.5 mg/ml                                                                              574 308582                                                                              347312    538 605                                  BDMQ       1 mg/ml                                                           93.                                                                              Agefloc A50                                                                               1 mg/ml 689 1015523                                                                             1225260   1474                                                                               1778                                 TBQ        1 mg/ml                                                           94.                                                                              Agefloc A50                                                                               0.5 mg/ml                                                                              693 309616                                                                              355165    447 513                                  TBQ        1 mg/ml                                                           95.                                                                              Agefloc A50HV                                                                             0.5 mg/ml                                                                              1265                                                                               1190551                                                                             1343823   941 1062                                 TBQ        1 mg/ml                                                           96.                                                                              Agefloc A50HV                                                                             0.5 mg/ml                                                                              769 284170                                                                              325540    370 423                                  TBQ        1 mg/ml                                                           97.                                                                              SDBS       0.5 mg/ml                                                                              725 758078                                                                              2071149   1046                                                                               2857                                 Tween-20   20 mg/ml                                                            BDMQ       1 mg/ml                                                           98.                                                                              SDBS       0.5 mg/ml                                                                              668 638270                                                                              1763156   955 2639                                 Triton X-100                                                                              1 mg/ml                                                             BDMQ       1 mg/ml                                                           99.                                                                              SDBS       0.5 mg/ml                                                                              1337                                                                               1021526                                                                             2582544   764 1932                                 CHAPS      1 mg/ml                                                             BDMQ       1 mg/ml                                                          100.                                                                              SDBS       0.5 mg/ml                                                                              1476                                                                               940142                                                                              2282165   637 1546                                 CHAPSO     1 mg/ml                                                             BDMQ       1 mg/ml                                                          101.                                                                              SDBS       0.5 mg/ml                                                                              1329                                                                               883203                                                                              2195667   665 1652                                 CTAB       0.05 mg/ml                                                          BDMQ       1 mg/ml                                                          102.                                                                              DAXAD      1 mg/ml 866 1059832                                                                             1225160   1224                                                                               1415                                 TBQ        1 mg/ml                                                          103.                                                                              DAXAD      0.5 mg/ml                                                                              802 771273                                                                              833134    962 1039                                 BDMQ/TBQ   1 mg/ml                                                             (20% BDMQ)                                                                  104.                                                                              DAXAD      0.5 mg/ml                                                                              510 301223                                                                              351656    591 690                                  BDMQ       1 mg/ml                                                          105.                                                                              DextranSul 0.5 mg/ml                                                                              306 37132                                                                               39345     121 129                                  BDMQ       0.1 mg/ml                                                        106.                                                                              SDS        0.6 mg/ml                                                                              961 772634                                                                              2258788   804 2350                                 Tween-20   1 mg/ml                                                             TBQ        1 mg/ml                                                          107.                                                                              SDS        0.6 mg/ml                                                                              1100                                                                               867025                                                                              2581024   788 2346                                 Triton X-100                                                                              1 mg/ml                                                             TBQ        1 mg/ml                                                          108.                                                                              SDS        0.6 mg/ml                                                                              721 592457                                                                              1575837   822 2186                                 Tween-20   1 mg/ml                                                             BDMQ       1 mg/ml                                                          109.                                                                              SDS        0.6 mg/ml                                                                              697 412432                                                                              1143747   592 1641                                 Triton X-100                                                                              1 mg/ml                                                             BDMQ       1 mg/ml                                                          110.                                                                              SDBS       0.1 mg/ml                                                                              2097                                                                               957195                                                                              2016212   456 962                                  Tween-20   1 mg/ml                                                             octyl-glucoside                                                                           1 mg/ml                                                             TBQ        1 mg/ml                                                          111.                                                                              SDBS       0.1 mg/ml                                                                              2276                                                                               924169                                                                              2002195   406 880                                  Triton X-100                                                                              1 mg/ml                                                             octyl-glucoside                                                                           1 mg/ml                                                             TBQ        1 mg/ml                                                          112.                                                                              SDBS       0.5 mg/ml                                                                              288 22720                                                                               56363     79  196                                  Tween-20   1 mg/ml                                                             CTAB       1 mg/ml                                                             TBQ        1 mg/ml                                                          113.                                                                              SDBS       0.5 mg/ml                                                                              291 17291                                                                               43494     59  149                                  Triton-X-100                                                                              1 mg/ml                                                             CTAB       1 mg/ml                                                             TBQ        1 mg/ml                                                          114.                                                                              SDBS       0.5 mg/ml                                                                              231 34158                                                                               101381    148 439                                  Tween-20   1 mg/ml                                                             BDMCAC     1 mg/ml                                                             TBQ        1 mg/ml                                                          115.                                                                              SDBS       0.5 mg/ml                                                                              233 31723                                                                               90256     136 387                                  Triton X-100                                                                              1 mg/ml                                                             BDMCAC     1 mg/ml                                                             TBQ        1 mg/ml                                                          116.                                                                              SDBS       0.1 mg/ml                                                                              878 1052025                                                                             2086077   1198                                                                               2376                                 Tween-20   1 mg/ml                                                             CTAB       0.1 mg/ml                                                           TBQ        1 mg/ml                                                          117.                                                                              SDBS       0.1 mg/ml                                                                              1009                                                                               1358360                                                                             2551569   1346                                                                               2529                                 Tween-20   1 mg/ml                                                             CTAB       0.01 mg/ml                                                          TBQ        1 mg/ml                                                          118.                                                                              CTAB       0.1 mg/ml                                                                              412 395868                                                                              714210    961 1734                                 Triton X-100                                                                              1 mg/ml                                                             TBQ        1 mg/ml                                                          119.                                                                              CTAB       0.01 mg/ml                                                                             632 727582                                                                              1089538   1151                                                                               1724                                 Triton X-100                                                                              1 mg/ml                                                             TBQ        1 mg/ml                                                          120.                                                                              octyl-glucoside                                                                           1 mg/ml 586 726013                                                                              836153    1239                                                                               1427                                 TBQ        1 mg/ml                                                          121.                                                                              Tween-20   1 mg/ml 550 371246                                                                              620097    675 1128                                 TBQ        1 mg/ml                                                          122.                                                                              Triton X-100                                                                              1 mg/ml 636 329832                                                                              570539    5.19                                                                               897                                  TBQ        1 mg/ml                                                          123.                                                                              CTAB       1 mg/ml 227 15217                                                                               26636     67  117                                  TBQ        1 mg/ml                                                          124.                                                                              Polyox(50K)                                                                               20 mg/ml                                                                               154 26179                                                                               47219     170 307                                  CTAB       0.25 mg/ml                                                       125.                                                                              Polyox(50K)                                                                               20 mg/ml                                                                               243 495112                                                                              723580    2037                                                                               2978                                 CTAB       0.25 mg/ml                                                          TBQ        1 mg/ml                                                          126.                                                                              Polyox(50K)                                                                               20 mg/ml                                                                               607 768812                                                                              930826    1267                                                                               1533                                 CTAB       0.025 mg/ml                                                         TBQ        1 mg/ml                                                          127.                                                                              Polyox(50K)                                                                               20 mg/ml                                                                               402 832297                                                                              962123    2070                                                                               2393                                 CTAB       0.0025 mg/ml                                                        TBQ        1 mg/ml                                                          128.                                                                              Polyox(50K)                                                                               0.5 mg/ml                                                                              391 970406                                                                              1102900   2482                                                                               2821                                 TBQ        1 mg/ml                                                          129.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               283 42628                                                                               77784     151 275                                  CTAB       0.25 mg/ml                                                       130.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               275 36981                                                                               44021     134 160                                  CTAB       0.025 mg/ml                                                      131.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               250 40563                                                                               45500     162 182                                  CTAB       0.0025 mg/ml                                                     132.                                                                              Polyox(500K)                                                                              1 mg/ml 397 1527695                                                                             1781760   3848                                                                               4488                                 CTAB       0.05 mg/ml                                                          TBQ        1 mg/ml                                                          133.                                                                              Polyox(500K)                                                                              1 mg/ml 455 1629263                                                                             1834806   3581                                                                               4033                                 CTAB       0.005 mg/ml                                                         TBQ        1 mg/ml                                                          134.                                                                              Polyox(500K)                                                                              1 mg/ml 514 1635987                                                                             1823578   3183                                                                               3548                                 CTAB       0.0005 mg/ml                                                        TBQ        1 mg/ml                                                          135.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               623 2505788                                                                             3072392   4022                                                                               4931                                 TBQ        1 mg/ml                                                          136.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               432 528833                                                                              653731                                                                              669867                                                                              1224                                                                               1513                                                                               1551                             BDMQ       1 mg/ml                                                          137.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               17614                                                                              2047964                                                                             4050329   116 230                                  Zelec DP   2.5 μl/ml                                                        Tween-20   0.625 mg/ml                                                         TBQ        1 mg/ml                                                          138.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               562 76352                                                                               229205    136 408                                  SDBS       0.5 mg/ml                                                           CTAB       0.75 mg/ml                                                          Tween-20   1 mg/ml                                                             TBQ        1 mg/ml                                                          139.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               2689                                                                               474537                                                                              1267603   176 471                                  SDBS       0.5 mg/ml                                                           CTAB       0.5 mg/ml                                                           Tween-20   1 mg/ml                                                             TBQ        1 mg/ml                                                          140.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               5476                                                                               675082                                                                              535300    123 280                                  SDBS       0.25 mg/ml                                                          CTAB       0.3 mg/ml                                                           Tween-20   1 mg/ml                                                             TBQ        1 mg/ml                                                          141.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               729 393791                                                                              507399    540 696                                  1M NaCl in DEA                                                                 TBQ        1 mg/ml                                                          142.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               714 444112                                                                              545624    622 764                                  0.5M NaCl in DEA                                                               TBQ        1 mg/ml                                                          143.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               803 271814                                                                              311794    338 388                                  1M NaCl (no DEA)                                                               TBQ        1 mg/ml                                                          144.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               723 316032                                                                              342073    437 473                                  0.5M NaCl (noDEA)                                                              TBQ        1 mg/ml                                                          145.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               810 1255809                                                                             2567260   1550                                                                               2388                                 SDBS       0.1 mg/ml                                                           Chaps      1 mg/ml                                                             Tween-20   1 mg/ml                                                             TBQ        1 mg/ml                                                          146.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               858 1529784                                                                             3035681   1783                                                                               2575                                 SDBS       0.1 mg/ml                                                           Chapso     1 mg/ml                                                             Tween-20   1 mg/ml                                                             TBQ        1 mg/ml                                                          147.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               1309                                                                               1581194                                                                             3072325   1208                                                                               1674                                 SDBS       0.1 mg/ml                                                           Chaps      1 mg/ml                                                             Triton-X100                                                                               1 mg/ml                                                             TBQ        1 mg/ml                                                          148.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               2593                                                                               1240847                                                                             2249328   479 591                                  SDBS       0.05 mg/ml                                                          Chapso     0.5 mg/ml                                                           Tween-20   1 mg/ml                                                             TBQ        1 mg/ml                                                          149.                                                                              Polybrane  0.5 mg/ml                                                                              425 979550                                                                              1210591   2305                                                                               2848                                 TBQ        1 mg/ml                                                          150.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               506 1106515                                                                             1378277   2187                                                                               2724                                 Polybrene  0.5 mg/ml                                                           TBQ        1 mg/ml                                                          151.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               765 995361                                                                              1202025   1301                                                                               1571                                 CHAPS      0.5 mg/ml                                                           Polybrene  0.5 mg/ml                                                           TBQ        1 mg/ml                                                          152.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               2024                                                                               1149546                                                                             1385744   568 685                                  CHAPSO     0.5 mg/ml                                                           Polybrene  0.5 mg/ml                                                           TBQ        1 mg/ml                                                          153.                                                                              Polybrene  0.5 mg/ml                                                                              567 1144898                                                                             1337005   2019                                                                               2358                                 CHAPS      0.5 mg/ml                                                           TBQ        1 mg/ml                                                          154.                                                                              Polybrene  0.5 mg/ml                                                                              665 1240889                                                                             1442628   1866                                                                               2169                                 CHAPSO     0.5 mg/ml                                                           TBQ        1 mg/ml                                                          155.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               573 766172                                                                              942897    1337                                                                               1646                                 DEA/Carb buffer 9.5                                                            TBQ        1 mg/ml                                                          156.                                                                              TBQ        1 mg/ml 466 320952                                                                              371408    689 797                                  DEA/Carb buffer 9.5                                                         157.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               531 1996396                                                                             2509803   3760                                                                               4727                                 TBQ        0.5 mg/ml                                                        158.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               482 1649504                                                                             2021238   3422                                                                               4193                                 octyl-glucoside                                                                           1 mg/ml                                                             TBQ        1 mg/ml                                                          159.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               262 35061                                                                               36818     134 141                                  octyl-glucoside                                                                           5 mg/ml                                                          160.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               604 1729994                                                                             2149082   2864                                                                               3558                                 phenyl-glucoside                                                                          5 mg/ml                                                             TBQ        1 mg/ml                                                          161.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               268 30705                                                                               33366     150 124                                  phenyl-glucoside                                                                          5 mg/ml                                                          162.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               562 1372766                                                                             2935468   2443                                                                               5223                                 octyl-glucoside                                                                           2.5 mg/ml                                                           SDBS       0.25 mg/ml                                                          Tween-20   1 mg/ml                                                             TBQ        1 mg/ml                                                          163.                                                                              Polyox(500K)                                                                              20 mg/ml                                                                               632 1197951                                                                             2459440   1895                                                                               3892                                 phenyl-glucoside                                                                          2.5 mg/ml                                                           SDBS       0.25 mg/ml                                                          Tween 20   1 mg/ml                                                             TBQ        1 mg/ml                                                          164.                                                                              SDBS       0.1 mg/ml                                                                              413 1457360                                                                             2768643                                                                             3541193                                                                             3529                                                                               6704                                                                               8574                             Tween-20   1 mg/ml                                                             TBQ        1 mg/ml                                                          165.                                                                              SDBS       0.1 mg/ml                                                                              414 1353062                                                                             2649803                                                                             3601796                                                                             3268                                                                               6400                                                                               8700                             Tween-20   1 mg/ml                                                             TBQ        0.5 mg/ml                                                        166.                                                                              SBS        1 mg/ml 318 405862                                                                              478723                                                                              473862                                                                              1276                                                                               1505                                                                               1490                             BDMQ       1 mg/ml                                                          167.                                                                              SBS        0.5 mg/ml                                                                              341 950755                                                                              1115291                                                                             1107561                                                                             2788                                                                               3271                                                                               3248                             TBQ        1 mg/ml                                                          __________________________________________________________________________

                                      TABLE 7                                      __________________________________________________________________________     AMPPD                                                                                                  Signal        S/N                                      Additive    Concentration                                                                          Noise                                                                              10 min                                                                              30 min                                                                              60 min                                                                             10 min                                                                             30 min                                                                             60 min                           __________________________________________________________________________     1. none             601 15434                                                                               22361    26  37                                   2. BDMQ     1 mg/ml 1021                                                                               239457                                                                              392753   250 410                                  3. TBQ      1 mg/ml 1940                                                                               5429838                                                                             953548   311 819                                  4. TBQ/THQ  1 mg/ml 10112                                                                              3909328                                                                             7854723  387 777                                  5. BDMCAC   0.5 mg/ml                                                                              539 19978                                                                               59936    39  118                                  6. THQ monomer                                                                             5 mg/ml 611 38535                                                                               92199    58  140                                  7. Andogen 464                                                                             1 mg/ml 1019                                                                               232240                                                                              713917   253 779                                  8. Andogen 464                                                                             5 mg/ml 447 19223                                                                               63303    43  214                                     Triton X-100                                                                            10 mg/ml                                                           9. Andongen 464                                                                            5 mg/ml 784 49888                                                                               167538   64  214                                     Triton X-100                                                                            1 mg/ml                                                            10.                                                                               Andogen 464                                                                             5 mg/ml 493 32050                                                                               107977   65  219                                     Tween-20 10 mg/ml                                                              Andogen 464                                                                             1 mg/ml 718 39265                                                                               131675   55  183                                     Tween-20 1 mg/ml                                                               BDMTDAC  0.1 mg/ml                                                                              1213                                                                               94008                                                                               210047   78  173                                     BDMQ     1 mg/ml                                                               BDMDAB   0.1 mg/ml                                                                              1366                                                                               156795                                                                              273187   115 200                                     BDMQ     1 mg/ml                                                               Zonyl Surf.                                                                             0.1 mg/ml                                                                              1597                                                                               170683                                                                              283296   107 177                                     BDMQ     1 mg/ml                                                               BDMTDAC  0.1 mg/ml                                                                              752 186443                                                                              432829   248 576                                     TBQ      1 mg/ml                                                               BDMDAB   0.1 mg/ml                                                                              1000                                                                               379583                                                                              717087   380 717                                     TBQ      1 mg/ml                                                               Zonyl Surf.                                                                             0.1 mg/ml                                                                              1108                                                                               393546                                                                              734506   355 663                                     TBQ      1 mg/ml                                                               octyl-glucoside                                                                         0.1 mg/ml                                                                              1474                                                                               167319                                                                              316876   35  68                                      BDMQ     1 mg/ml                                                               phenyl-glucoside                                                                        0.1 mg/ml                                                                              1663                                                                               168296                                                                              31431    101 189                                     BDMQ     1 mg/ml                                                            20.                                                                               octyl-glucoside                                                                         1 mg/ml 359 16800                                                                               41762    47  116                                     BDMDAB   1 mg/ml                                                               BDMQ     1 mg/ml                                                               octyl-glucoside                                                                         0.5 mg/ml                                                                              1000                                                                               339007                                                                              648072   339 648                                     TBQ      1 mg/ml                                                               phenyl-glucoside                                                                        0.5 mg/ml                                                                              1221                                                                               269016                                                                              545848   220 439                                     TBQ      1 mg/ml                                                               HDTBPB   0.5 mg/ml                                                                              1412                                                                               36919                                                                               102172   26  72                                      Andogen 464                                                                             1 mg/ml 3919                                                                               223888                                                                              682817   57  174                                     phenyl-glucoside                                                                        1 mg/ml                                                               HDTBPB   0.5 mg/ml                                                                              640 40379                                                                               124470   63  195                                     phenyl-glucoside                                                                        1 mg/ml                                                               Nonidet P-40                                                                            0.1 mg/ml                                                                              1143                                                                               112668                                                                              252316   99  221                                     BDMQ     1 mg/ml                                                               Tween-80 0.5 mg/ml                                                                              844 87136                                                                               165285   193 196                                     BDMQ     1 mg/ml                                                               PO23LE   0.1 mg/ml                                                                              1980                                                                               130369                                                                              261388   66  132                                     BDMQ     1 mg/ml                                                               Ipegal   0.5 mg/ml                                                                              822 116303                                                                              231011   142 281                                     BDMQ     1 mg/ml                                                            30.                                                                               HDTBPB   0.1 mg/ml                                                                              1167                                                                               35861                                                                               90738    31  78                                      BDMQ     1 mg/ml                                                               β-cyclodex.                                                                        0.5 mg/ml                                                                              451 46605                                                                               101249   103 224                                     BDMQ     1 mg/ml                                                               FC-135   0.5 mg/ml                                                                              420 66398                                                                               133292   158 317                                     BDMQ     1 mg/ml                                                               BOP      1 mg/ml 3327                                                                               464178                                                                              1059036  140 318                                     BDMQ     1 mg/ml                                                               PPG      1 mg/ml 803 25376                                                                               411277   316 512                                     BDMQ     1 mg/ml                                                               PEI      0.1 mg/ml                                                                              771 265606                                                                              446988   345 580                                     BDMQ     1 mg/ml                                                               P-DMDMPC 0.5 mg/ml                                                                              1031                                                                               286017                                                                              489468   277 475                                     BDMQ     1 mg/ml                                                               Avitex DN                                                                               5 mg/ml 372 33046                                                                               67404    59  181                                     Avitex DN                                                                               0.1 mg/ml                                                                              687 251317                                                                              418395   366 609                                     BDMQ     1 mg/ml                                                               Avitex E 0.5 mg/ml                                                                              674 273361                                                                              442352   406 656                                     BDMQ     1 mg/ml                                                            40.                                                                               DMQ-TEQ  5 mg/ml 826 198197                                                                              445457   240 539                                     TBQ/THQ  1 mg/ml 20254                                                                              2910352                                                                             6446798  144 318                                     (35 mole % THQ)                                                                BDMQ     1 mg/ml                                                               DMQ-TEQ  5 mg/ml 706 226048                                                                              502481   320 712                                     BDMQ     1 mg/ml                                                               TBQ/THQ  1 mg/ml 20339                                                                              2415719                                                                             5398310  119 265                                     (35 mole % THQ)                                                                TBQ      1 mg/ml                                                               DMQ-TEQ  1 mg/ml 893 370453                                                                              735995   415 824                                     TBQ      1 mg/ml                                                               Zelec DP 2.5 μl/ml                                                                           1916                                                                               340564                                                                              8009917  178 423                                     phenyl-glucoside                                                                        1 mg/ml                                                               BDMQ     1 mg/ml                                                               Zelec DP 2.5 μl/ml                                                                           2244                                                                               386368                                                                              1068947  172 476                                     phenyl-glucoside                                                                        1 mg/ml                                                               TBQ      1 mg/ml                                                               PVA      1 mg/ml 365 395636                                                                              476147   1084                                                                               1305                                    BDMQ     1 mg/ml                                                            __________________________________________________________________________

Applicants have endeavored to illustrate their invention by extensive embodiment of possible combinations. Nonetheless, it is recognized that the possible combinations are endless, and cannot be exhaustively embodied. Given the above teaching, those of ordinary skill in the art will arrive at enhancement agents and additives not specifically exemplified in the foregoing application. The examples are not intended to be limiting, and the identification of other combinations, given the foregoing disclosure, is well within the skill of those practicing this technology without undue experimentation. Such combinations are intended to be within the scope of the invention, save as expressly limited or excluded by the claims set forth below. 

We claim:
 1. In a method for determining the presence or concentration of an analyte in an aqueous sample comprising admixing said sample with an enzyme complex capable of binding to said analyte, removing all unbound enzyme complex and adding to said treated sample a 1,2-dioxetane which is caused to decompose with the release of chemiluminescence when contacted by said enzyme thereby forming a 1,2-dioxetane oxyanion and excited state emitters, and wherein the amount of chemiluminescence is monitored to determine the presence or concentration of said analyte, the improvement comprising adding to said aqueous sample 0.005%-50%, weight by volume of the sample, poly(vinylbenzyltributylammonium chloride) (TBQ).
 2. The method of claim 1, wherein said method further comprises the addition to said sample of 0.005%-50% weight by volume of said sample of an enhancement additive which improves the ability of said poly(vinylbenzyltributylammonium chloride) TBQ to form hydrophobic regions in said aqueous sample in which said 1,2-dioxetane oxyanion and its decomposition products can be sequestered.
 3. The method of claim 2, wherein more than one said additive is added to said sample.
 4. The method of claim 2, wherein said additive is selected from the group consisting of surfactants, negatively charged salts, an alcohol, turpentine solvents, and water-soluble polymers.
 5. The method of claim 2, wherein said additive comprises Zelec DP.
 6. A method for determining the presence or concentration of an analyte in an aqueous sample comprising admixing said sample with an enzyme complex capable of stably binding to said analyte, removing all unbound enzyme complex and adding to said sample a 1,2-dioxetane which is caused to decompose with the release of chemiluminescence upon contact with said enzyme and wherein the amount of chemiluminescence is monitored to determine the presence or concentration of said analyte, the improvement comprising adding to said sample(a) an enhancement agent comprising a water-soluble polymeric quaternary onium salt in an amount of 0.005%-50% weight by volume of said sample, which, when added to said sample, results in the generation of greater chemiluminescence due to the decomposition of said 1,2-dioxetane than in the absence of said water-soluble polymeric quaternary onium salt, and (b) 0.005%-50% weight based on volume of said sample, of an enhancement additive which improves the ability of said polymeric quaternary onium salt to form hydrophobic regions in said aqueous sample in which said 1,2-dioxetane and its chemiluminescent decomposition products can be sequestered.
 7. The method of claim 6, wherein said enhancement agent is prepared from monomers selected from the group consisting of quaternary ammonium salts, quaternary sulfonium salts, quaternary phosphonium salts and mixtures thereof.
 8. The method of claim 6, wherein said enhancement agent is a polymeric quaternary ammonium salt, a polymeric quaternary sulfonium salt, a polymeric quaternary phosphonium salt or copolymers thereof.
 9. The method of claim 8, wherein said enhancement agent is selected from the group consisting of poly(vinylbenzyldimethylbenzylammonium chloride) (BDMQ), poly(vinylbenzyltrimethylammonium chloride) (TMQ), poly(vinylbenzyltributylammonium chloride) (TBQ) and copolymers thereof.
 10. The method of claim 6, wherein said process further comprises the addition of a second enhancement additive which is a detergent.
 11. A method of conducting an assay for the presence or concentration of analyte in a aqueous sample, comprising:admixing an enzyme complex with said sample, which enzyme complex will stably bind to said analyte in said sample upon said admixture, removing unbound enzyme complex present in said sample after said admixture, adding to said samples 0.005%-50%, weight by volume of said sample, of poly(vinylbenzyltributylammonum chloride) (TBQ), adding a 1,2-dioxetane to said sample, which dioxetane is caused by the enzyme of said enzyme complex to decompose into a decomposition product which chemiluminesces, and measuring the amount of chemiluminescence obtained, wherein said chemiluminescence is indicative of said presence or concentration of said analyte.
 12. The method of claim 1, wherein said 1,2-dioxetane has the formula: ##STR4## Z=H, Cl, other halogens or alkoxy groups; R¹ is C₁ -C₂₀ alkyl or C₁₋₁₂ aryl or aralkyl;Y is phenyl or naphthyl; R² is meta-substituted or nonconjugated on Y with respect to the dioxetane, and is OX, wherein; X is an enzyme cleavable group which, when cleaved, leaves the dioxetane phenoxy or naphthoxy anion.
 13. A method of conducting an assay for the presence or concentration of an analyte in a aqueous sample, comprising:admixing an enzyme complex with said sample, which enzyme complex will stably bind to said analyte in said sample upon said admixture, removing unbound enzyme complex from said sample after said admixture, adding to said sample 0.005%-50% weight by volume of said sample volume, a water soluble polymeric quaternary onium salt enhancement agent together with 0.005%-50% weight by volume of said sample, of an enhancement additive which improves the ability of said enhancement agent to form hydrophobic regions in said sample and in which decomposition products of a 1,2-dioxetane added to said sample and caused to decompose by contact with the enzyme of said enzyme complex can be sequestered, provided the sum of said enhancement agent and enhancement additive does not exceed 50% weight by volume of the sample, adding said 1,2-dioxetane to said sample, said dioxetane being caused by the contact of the enzyme of said enzyme complex to decompose into said decomposition product which chemiluminesces, and measuring the amount of chemiluminescence obtained, wherein the amount of chemiluminescence observed in the presence of said enzyme complex is greater than the amount of chemiluminescence observed is greater than the amount of chemiluminescence observed in the absence of said enhancement additive, and wherein said chemiluminescnce is indicative of the presence or concentration of said analyte.
 14. The method of claim 13, wherein said enhancement agent is prepared from monomers selected from the group consisting of quaternary ammonium monomers, quaternary phosphonium monomers, quaternary sulfonium monomers and mixtures thereof.
 15. The method of claim 13, wherein said enhancement additive is selected from the group consisting of a detergent, a negatively charged salt, an alcohol or turpentine solvent, and a water soluble polymer.
 16. A kit for conducting a bioassay for the presence or concentration of an analyte in aqueous sample, comprising:an enzyme complex which will bind to said analyte in said sample upon admixture therewith, a 1,2-dioxetane which when contacted by the enzyme of said enzyme complex will be caused to decompose into a decomposition product which chemiluminesces, and TBQ.
 17. A kit for conducting a bioassay for the presence of concentration of an analyte in a sample, comprising:an enzyme complex which will bind to said analyte upon admixture with said sample, a 1,2-dioxetane which upon contact with the enzyme of said enzyme complex will be caused to decompose into a 1,2-dioxetane oxyanion and decomposition product which chemiluminesce, a polymeric quaternary onium salt and an enhancement additive which improves the ability of said polymeric quaternary onium salt to form hydrophobic regions in said sample in which said 1,2-dioxetane oxyanion and its decomposition product can be sequestered,.
 18. The kit of claim 17, wherein said enhancement additive is selected from the group consisting of a detergent, a negatively charged salt, an alcohol, turpentine solvent and a water-soluble polymer.
 19. The method of claim 1, wherein said dioxetane is the disodium salt of 3-(4-methoxyspiro[1,2-dioxetane-3,2' tricyclo[3.3.1.1.³,7 ]decan]-4-yl)phenyl phosphate (AMPPD) or chloroadamant-2'-ylidenemethoxyphenoxy phosphorylated dioxetane (CSPD).
 20. The method claim 13, wherein said dioxetane is AMPPD, CSPD or a mixture thereof, said enhancement agent is selected from the group consisting of TBQ, BDMQ, TMQ and mixtures thereof, and said enhancement additive is Zelec DP. 